Gel electrophoresis is a common procedure for the separation of biological molecules, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polypeptides and proteins. In gel electrophoresis, the molecules are separated into bands according to the rate at which an imposed electric field causes them to migrate through a filtering gel.
The basic apparatus used in this technique consists of a gel often enclosed in a glass tube or sandwiched as a slab between glass or plastic plates. The gel has an open molecular network structure, defining pores, which are saturated with an electrically conductive buffered solution of salts. These pores through the gel are large enough to admit passage of the migrating macromolecules.
The gel is placed in a chamber in contact with buffer solutions which make electrical contact between the gel and the cathode and anode of an electrical power supply. A sample containing the macromolecules and a tracking dye is placed on top of the gel. An electric potential is applied to the gel causing the sample macromolecules and tracking dye to migrate toward the bottom of the gel. The electrophoresis is halted just before the tracking dye reaches the end of the gel. The locations of the bands of separated macromolecules are then determined. By comparing the distance moved by particular bands in comparison to the tracking dye and macromolecules of known mobility, the mobility of other macromolecules can be determined. The size of the macromolecule can then be calculated or macromolecules of different sizes can be separated in the gel.
There are a wide range of gel-forming materials used for electrophoresis. Polyacrylamides, polymethacrylamides and other related polymers are preferred for separation of smaller molecular weight materials such as proteins, peptides and small nucleic acids. Conversely, agarose, cellulose acetate and starch are preferred for larger molecules. These gel materials are typically compatible with aqueous systems, though some are also compatible with non-aqueous solvents.
Formation of the gel material to the desired physical dimensions can be accomplished by varying techniques, depending on the material chosen. With agarose or gelatin the common method is to heat the polymer causing the material to go into solution. The solution can then be poured into a cast and allowed to polymerize by cooling. Alternatively, polyacrylamides, polymethacrylamides and other related polymers can be chemically polymerized by various means including free radical induced polymerization with ammonium persulfate and tetramethylethylenediamine.
A common problem with gel electrophoresis is the difficulty of removing the protein, nucleic acid or other analyte of interest from the gel after it has been separated from other components. Because the gel matrix has very small pore sizes, large molecules do not easily diffuse out of the gel matrix after they have been drawn into the matrix through electromotive force. Proteins also do not diffuse into the gel matrix readily in the absence of electromotive force. The larger the molecular weight of the protein the more difficult it is to get the molecules into or out of the gel. Thus, techniques have been developed for preparing a gel for extraction or introduction of molecules out of or into the gel.
The state of the art includes a procedure for gel subdivision using a sieve. Christoph Eckerskom and Rudolf Grimm describe the use of a stainless steel sieve placed in the end of a syringe barrel for subdividing gels. Eckerskorn and Grimm attribute their technique to J. Heukeshoven and R. Dernick, as described in B. Radola (ed.), Electrophoresis Forum ′91, Technical University, Munich 1991, pp. 501-506. In another article, J. Lila Castellanos-Serra, et al., Electrophoresis 1999; 20: 732-737, a stainless steel sieve screen is used for subdividing gels to remove proteins for further analysis. This article attributes the idea to Eckerskorn and Grimm and to Heukeshoven and Dernick. Castellanos-Serra et al. placed a piece of stainless sieve screen in the narrow end of a syringe barrel and used the pressure of the plunger to force the gel through the screen.
However, this approach has significant disadvantages. First, the syringe is costly, especially if there are large numbers of gels to be processed simultaneously. This cost arises in part because the syringe comprises several parts including a barrel, plunger and gasket. Second, the user must manually force the gel through the mesh with the action of a plunger. This technique is significantly labor intensive and is not amenable to automation. Third, the plunger does not advance all of the gel cleanly through the mesh, because the force applied by the plunger stops at the top of the mesh.
Others have demonstrated that centrifugal force can be useful in forcing materials through a barrier for various purposes, e.g. a filtration membrane for separation. For example, U.S. Pat. No. 3,583,627 to Wilson describes concentrating a large molecular weight substance in solution by fixing a filter into the end of the upper of two nested tubes and spinning the tubes to force the solvent through the filter while retaining the macromolecules. There are numerous examples of using this basic principle to concentrate macromolecules, for example U.S. Pat. No. 4,632,761 Bower et al. These devices and methods, however, are designed for filtration of solutions and are not suitable for cutting or subdividing a semi-solid gel or other substance.
Additionally, Millipore Corporation of Bedford, Mass. currently sells Product No. 42600 “Ultrafree DA for DNA extraction,” for the subdivision of gel fragments. This product also suffers from several drawbacks. First, it uses a nested tube set, the upper tube having in its base plastic projections molded in place that are supposed to subdivide a gel when the tube set is spun. However, in practice, these do not work well because the resulting subdivided gel has large, inconsistently-sized pieces of gel leading to inefficient and unreliable extraction. Second, the Millipore device is recommended primarily for agarose gels and may be used on polyacrylamide gels, but only with a maximum polymer concentration of 10% by weight. Gels having a polymer concentration less than 10% by weight are usually unable to efficiently separate very low molecular weight peptides and proteins, which often require 12%, 15%, 18% or a higher percentage of polymer concentration by weight.
There is, therefore, a need for a method of easily, efficiently, reliably and inexpensively subdividing a gel to facilitate the extraction of various molecules from the gel, or conversely, the introduction of molecules into the gel. In particular, as more and more analytes become available for study, e.g. through the Human Genome Project and follow-on projects to identify genes and express gene products, there is a need to perform such extractions or introductions in high-throughput and automated formats.
Although much of the description of the invention is related to removing proteins from gels, it is also often of interest to remove other molecules from gels. A person of ordinary skill in the art would readily apply the techniques described herein to other molecules commonly subjected to electrophoresis, e.g. nucleic acids such as DNA or RNA.